Ionic modulation of triethyllead neurotoxicity in cerebellar granule cell culture.
While the molecular mechanism underlying triethyllead (TEL) neurotoxicity is unknown, we hypothesize that triethyllead mediates an accelerated Cl-/OH exchange across neuronal membranes leading to prolonged depolarization and neuronal cell injury. As a test of this hypothesis we have investigated the effect of external ion modulation on triethyllead neurotoxicity in cerebellar granule cell culture. Cultures were prepared from neonatal rats and used 10-20 days in vitro. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release and trypan blue exclusion. A slow, dose-dependent (1-30 microM TEL) release of LDH occurred after a variable latent period dependent upon [TEL]. External replacement of [Cl-]e by Na isothionate dramatically shifted the dose response curve to the left reflecting an accelerated stimulation of LDH release, while replacement of extracellular [Na+]e with equimolar choline chloride had a minimal protective effect. Similarly, high [Mg2+]e or low [Ca2+]e did not protect or potentiate TEL cytotoxicity. The low [Cl-]e accelerated TEL cytotoxicity was dependent on medium pH: alkaline pH potentiated the cytotoxicity. Low [Cl-]e had no significant effect on culture ATP over 5 hrs. ATP reduction was markedly stimulated by TEL in low Cl- medium in contrast to the minimal decline in [ATP] in the control medium. The reduction of ATP in the low [Cl-]e medium occurred prior to LDH or trypan blue staining release confirming that such reduction in [ATP] was not secondary to cell damage. Substituting K sulfate or Na sulfate for the Cl(-)-free medium revealed marked loss of ATP without LDH release in control and TEL supplemented cultures. These observations provide supporting evidence for the role of an abnormal Cl- flux in mediating TEL-induced neurotoxic injury. Specifically, the membrane depolarization is proportional to the gradient imposed by Cl- efflux/OH influx, stimulated by low [Cl-]e. The rapid loss in ATP appeared early, was not a secondary reflection of neuronal damage but a result of a combination of increased ion flux at the plasma membrane, stimulation of Na+/K+ ATPase and direct TEL-induced inhibition of mitochondrial oxidative phosphorylation.
Verity
M A
MA
Department of Pathology (Neuropathology), University of California School of Medicine, Los Angeles 90024-1732.
Sarafian
T S
TS
Guerra
W
W
Ettinger
A
A
Sharp
J
J
D000255:Adenosine Triphosphate / Q000032:analysis; D000818:Animals; D002118:Calcium / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D002712:Chlorides / Q000502:physiology; D007477:Ions; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008274:Magnesium / Q000502:physiology; D009419:Nerve Tissue Proteins / Q000032:analysis; D009942:Organometallic Compounds / Q000633:toxicity*; D011190:Potassium Cyanide / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D012964:Sodium / Q000502:physiology
D002712:Chlorides; D007477:Ions; D009419:Nerve Tissue Proteins; D009942:Organometallic Compounds; C010360:triethyllead; D000255:Adenosine Triphosphate; D012964:Sodium; D007770:L-Lactate Dehydrogenase; D008274:Magnesium; D011190:Potassium Cyanide; D002118:Calcium